Amyotrophic lateral sclerosis (ALS) and frontotemperal lobar degeneration (FTLD) are debilitating neurological disorders that cause extreme suffering to patients and caregivers alike. Affected neurons in the spinal cord and brain of patients with ALS and FTLD are characterized for many individuals by having too much ubiquinylated and misfolded inclusions of cytosolic trans-activating response (TAR) DNA binding protein TDP-43. It is estimated that half of FTLD patients have associated TDP-43 pathology, making TDP-43-associated FTLD the single largest subtype. TDP-43 is also a key component of the ubiquitinated inclusions found in the cytosol of most patients with ALS, especially sporadic ALS (sALS, 85-90% of patients). In addition, numerous mutations in TDP-43, particularly in the glycine-rich C-terminal domain, are linked to familial ALS (fALS). The translational research effort described in this grant application seeks to identify selective radiotracers that image TDP-43 in real time in the brain or spinal cord of relevant patient via positron emission tomography (PET). Unlike for amyloid (e.g. florbetapir) and tau, no TDP-43 radiotracers have been reported to date. PET ligands for ALS and FTLD are expected to provide early and more accurate diagnosis of disease, help to monitor the progression of disease over time, and evaluate whether various therapeutic treatments are having a positive effect in individual patients. We have discovered new small-molecule probes that bind to TDP-43 using an alpha-screen assay that we developed, and here propose to further refine and validate these as radiotracers, including in animal models such as transgenic mice and normal non-human primates. Aim 1 is to obtain more potent TDP-43 binders as candidates for 18F or 11C hot ligand synthesis, by conducting iterative SAR development preparing ~200-250 new chemical entities (NCEs) to obtain small molecule candidates that bind to TDP-43 with PET-suitable biophysical properties, using modern methods of medicinal chemistry, structure-based design, pharmacophore development and synthetic chemistry. Biochemical characterization will use our alpha-screen assay and evaluation of binding to pathologically-relevant misfolded TDP-43. ADME characterization will ensure that the biophysical properties of the top leads selected are amenable for PET. In Aim 2, we will prepare radiolabeled TDP-43 binding ligands suitable for in vivo imaging based on top Aim 1 leads, an area of expertise for which Marty Pomper, Johns Hopkins, key personnel on the application, has considerable experience. Finally, in Aim 3, we seek to validate one or more TDP-43 PET ligands using ex vivo and in vivo methods including in vivo characterization in TDP-43 transgenic mice, TDP-43-?NLS mice and normal non-human primates, with a desired outcome of >80% specific TDP-43 blockade. It is expected that at the end of this two year funding period we will have in hand at least one compound en route to an IND application. An example of commercial use would be to confirm a TDP-43-based diagnosis for dementia caused by FTLD, when compared to the amyloid-associated Alzheimer's disease.